DESCRIPTION: This study extends previous investigations of DNA diagnostic approaches for the opportunistically pathogenic protist Acanthamoeba, the causative agent of the sight-threatening disease Acanthamoeba keratitis (AK). Prior work used nucleotide sequences of the small subunit ribosomal RNA (ssu rDNA) genes from the nucleus and mitochondria to determine the phylogenetic relationships of Acanthamoeba species/strains. These studies showed that the taxonomy of Acanthamoeba could be represented more reliably by "sequence types" (or genotypes) rather than by traditional approaches based on morphological characters. Importantly, results indicated that the vast majority of AK cases are associated with a specific Acanthamoeba genotype: T4. We have now identified genotypes associated with AK (AKpath), genotypes associated with other disease manifestations such as granulomatous amebic encephalitis (GAE) (NAKpath), and genotypes from environmental samples not found in infections (NONpath). Further, phylogenetic structure was observed within genotype T4, a genotype found in AK infections, non-AK infections, and the environment. To extend previous studies, we propose to identify protein-coding gene alleles that may be unique to the pathogenic genotypes of Acanthamoeba. First, we will identify sequence variation in three protein-coding genes (profilin [an actin binding protein], a serine protease-like enzyme, and actophorin [an actin depolymerizing factor]) that may be involved in the pathogenicity of the amoeba. Using known DNA sequences of these genes, PCR primers have been constructed to obtain DNA products from Acanthamoeba genotypes. The sequences of the products will be determined, and comparative analyses of variability will be performed. Allelic differences in genes potentially involved in pathogenicity will be correlated with the classification of pathogenic and non-pathogenic genotypes. The genes also represent an independent test of Acanthamoeba phylogenies that have been based on non-coding ssu rDNA genes. Preliminary results for one of these genes, profilin, supports its use in these studies. Second, we will determine the DNA sequence of a large portion (about 12kb) of the Acanthamoeba mitochondrial genome (designated mt-l) for different genotypes. Mitochondrial genes have been shown to play roles in pathogenicity in Acanthamoeba, and other organisms. First, we will examine variation in two genes (cytochrome oxidase I and II) located on this mitochondrial fragment. Comparative genomic analyses of the mt-l region will be performed for AKpath, NAKpath, and NONpath Acanthamoeba genotypes to test if sequence differences, or allelic variation of mitochondrial genes are correlated with pathogenicity. Next, other genes in the mt-l region will be analyzed. Since previous work has shown that the mitochondrial ssu rDNA distinguishes sub-lineages within the genotype associated with AK, the mt-l sequence from sub-lineages of T4 also will be determined for comparative analyses. Preliminary results show that this region can be amplified in disparate genotypes. Further, these results may provide potential targets for future treatment, since products of these genes may be antibiotic targets.